Cells are generally impermeable to macromolecules such as proteins, nucleic acids, lipids, carbohydrates and other chemical compounds. Methods for efficient translocation of these agents across biological membranes are thus useful for both therapeutic and research applications. However, existing methods of intracellular delivery, such as electroporation, liposome fusion, gene gun particle bombardment, DEAE-dextran transfection, recombinant viral infection, and direct microinjection are often inefficient and/or toxic to cells. Accordingly, there is a continuing need for improved intracellular delivery methods, including methods that can be used for in vivo delivery of agents such as proteins, nucleic acids, lipids, carbohydrates, small molecules and other chemical compounds.
Studies have identified polypeptide sequences known as protein transduction domains (PTDs) that may serve this need. PTDs, also known as “cell penetrating peptides” (CPPs), possess the ability to translocate across biological membranes. PTDs are relatively short amino acid sequences that can be linked to a cargo moiety, allowing transport of the cargo moiety across a biological membrane, such as a cell membrane, an organelle membrane, and/or a nuclear membrane. Cargo moieties can include, for example, small molecules, macromolecules, lipids, liposomes, carbohydrates, proteins, or nucleic acids. While the mechanism of translocation remains unclear, some studies suggest that the mechanism is energy-independent and non-receptor-mediated (Derossi et al., J. Biol. Chem. 269:10444-50 (1994); Derossi et al., J. Biol. Chem. 271:18188-93 (1996); Vives et al., J. Biol. Chem. 272:16010-7 (1997); Nagahara et al., Nat. Med. 4:1449-52 (1998); Schwarze et al., Trends Cell Biol. 10:290-5 (2000)). Other studies suggest that PTDs operate through pinocytotic mechanisms (Leifert et al., Gene Ther. 9:1422-8 (2002); Fittipaldi et al., J. Biol. Chem. 278:34141-9 (2003); Whitton and Whitton, Mol. Ther. 8:13-20 (2003); Lundberg et al., Mol. Ther. 8:143-50 (2003); Richard et al., J. Biol. Chem. 278:585-90 (2003); Vives et al., Curr. Protein Pept. Sci. 4:125-32 (2003)).
PTDs are typically cationic in nature, often comprising, for example, multiple arginines or lysines. Naturally occurring PTDs have been derived from proteins which can efficiently pass through biological membranes. The best characterized of these PTDs are derived from the Drosophila homeoprotein antennapedia transcription protein (AntHD) (Joliot et al., New Biol. 3:1121-34 (1991); Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-8 (1991); Le Roux et al., Proc. Natl. Acad. Sci. USA, 90:9120-4 (1993); Derossi et al., Trends Cell Biol. 8:84-87 (1998)), the herpes simplex virus structural protein VP22 (Elliott and O'Hare, Cell 88:223-33 (1997); Elliot et al., Cell 88:223-233 (1997)) and the HIV-1 transcriptional activator TAT protein (Green and Loewenstein, Cell 55:1179-1188 (1988); Frankel and Pabo, Cell 55:1189-1193 (1988); U.S. Pat. Nos. 5,804,604; 5,747,641; 5,674,980; 5,670,617; and 5,652,122). Recent studies have also identified other PTDs, such as Mph-1 (U.S. Patent Application No. 20060148060); Sim-2 (Chrast et al., Genome Res. 7, 615-624 (1997)); and Pep-1 and Pep-2 (Morris et al., Nat. Biotech. 19:1173-1175 (2001)), among others. In addition, studies have isolated artificial PTDs and PTDs selected from random libraries (see, e.g., Joliot and Prochiantz, Nat. Cell Biol. 6(3):189-96 (2004); Zhao and Weissleder, Med. Res. Rev. 24(1):1-12 (2003); Saalik et al., Bioconj. Chem. 15:1246-1253 (2004)).
PTD-mediated translocation appears to circumvent many of the problems and limitations associated with other techniques of intracellular delivery. This method of transduction appears to work on all cell types, and has been shown to efficiently transduce up to 100% of cells in culture with no apparent toxicity (Nagahara et al., Nat. Med. 4:1449-52 (1998)). PTDs have been used successfully to induce the intracellular uptake of full-length proteins (Nagahara et al., Nat. Med. 4:1449-52 (1998)), DNA, antisense oligonucleotides (Astriab-Fisher et al., Pharm. Res., 19:744-54 (2002)), small molecules (Polyakov et al., Bioconjug. Chem. 11:762-71 (2000)) and even inorganic iron particles (Dodd et al., J. Immunol. Methods 256:89-105 (2001); Wunderbaldinger et al., Bioconjug. Chem. 13:264-8 (2002); Lewin et al., Nat. Biotechnol. 18:410-4 (2000); Josephson et al., Bioconjug. Chem. 10:186-91 (1999)) suggesting that there is no apparent limit to the size of molecules that can be translocated.
Due to the many advantages of PTD-mediated intracellular delivery, there is a continuing need in the art to identify and characterize new PTDs. It would be useful to have new PTDs capable of efficiently and effectively translocating a variety of different types of cargo moieties across cellular membranes and thus useful for a multitude of therapeutic, diagnostic and research methods which require targeted delivery of cargo moieties into a cell or into one or more specific locations within a cell. Further, it would be especially useful to identify PTDs that can cross not only the outer cell membrane, but also the nuclear membrane. Such PTDs would be useful, for example, for gene therapy applications, or for in vitro or in vivo transfection of cells.
SLPI is a serine protease inhibitor that plays an important role in protection of the mucosal epidermis and skin (Thompson et al., Proc. Natl. Acad. Sci. USA 83:6692-6696 (1986); Franken et al., J. Histochem. Cytochem. 37:493-498 (1989); Masuda et al., British J. Pharmacol. 115:883-888 (1995)). SLPI is a potent inhibitor of leukocyte serine proteases such as elastase and cathepsin G from neutrophils, and chymase and tryptase from mast cells, as well as trypsin and chymotrypsin from pancreatic acinar cells (Jin et al., Cell 88:417-26 (1997) and references cited therein; Grüter et al., EMBO 7:345-51 (1988)). Studies suggest that SLPI also functions in wound healing (Zhu et al., Cell 111:867-878 (2002)) and epithelial proliferation (Zhang et al., J. Biol. Chem. 277:29999-30009 (2002)), and has antibacterial, antiviral and anti-inflammatory properties (McNeely et al., J. Clin. Invest. 96:456-464 (1995); Song et al., J. Exp. Med. 190:535-542 (1999); Hiemstra et al., Curr. Pharm. Des. 10:2891-2905 (2004)).
Human SLPI is a nonglycosylated, 11.7 kD protein consisting of a single 107 amino acid polypeptide chain. SLPI comprises two cysteine-rich domains with a protease inhibitory site situated at leucine 72 in the carboxy-terminal domain. SLPI is found in parotid saliva, and in seminal plasma, cervical, nasal, and bronchial mucus. In human epithelial cells, SLPI is constitutively expressed and its expression is increased by phorbol ester, TNF-α, and LPS at supraphysiologic concentrations, as well as by synergistic combinations of elastase and corticosteroids (Jin et al., supra).
In the immune system, studies show SLPI to be a lipopolysaccharide (LPS)-induced, IFNγ-suppressible phagocyte product that inhibits LPS responses. SLPI binds to the membrane of human macrophages through annexin II (Jin et al., supra; Ma et al., J. Exp. Med. 200:1337-46 (2004)). SLPI binds to NF-κB binding sites in the promoter regions of the IL-8 and TNF-α genes in monocytes and inhibits the expression of those genes (Taggart et al., J. Exp. Med. 202:1659-68 (2005) and references cited therein). It has been suggested that the anti-inflammatory function of SLPI arises from such gene inhibition.
Recent studies suggest that SLPI may play a neuroprotective role in focal stroke because of rapid inhibition of activated proteases and its suppression in inflammatory response mediated by leukocytes (e.g., neutrophils and macrophages), which contributes to ischemic brain injury (Taggart et al., supra; Ilzecka et al., Cerebrovascular Diseases 13:38-42 (2002)). Elevated levels of serum SLPI are also observed in human stroke patients (Ilzecka et al., supra).
We previously discovered that secretory leukocyte protease inhibitor (“SLPI”) overcomes the inhibitory effect of myelin inhibitors on nerve fiber growth and promotes neuronal (e.g., axonal) regeneration. Before that discovery, it was not known that SLPI possesses neurostimulatory function. See PCT/US2007/008270, filed Mar. 30, 2007, which is incorporated by reference herein in its entirety.
Upon incubation with peripheral blood monocytes and the U937 monocytic cell line, SLPI has been shown to enter cells, becoming rapidly localized to the cytoplasm and nucleus (Taggart et al., supra). But the portions of SLPI necessary and sufficient for cellular localization of SLPI were not known. Nor was it known whether part or all of the amino acid sequences of SLPI could be linked to a cargo moiety and co-transport that moiety across one or more biological membranes.